We investigate the molecular mechanisms by which transmembrane proteins (referred to as cargo) are sorted to different compartments of the endomembrane system in eukaryotic cells. This system comprises an array of membrane-enclosed organelles including the endoplasmic reticulum (ER), the Golgi apparatus, the trans-Golgi network (TGN), endosomes, lysosomes, lysosome-related organelles (LROs) (e.g., melanosomes), and different domains of the plasma membrane in polarized cells (e.g., epithelial cells and neurons). Transport of cargo between these compartments is mediated by carrier vesicles or tubules that bud from a donor compartment, translocate through the cytoplasm, and eventually fuse with an acceptor compartment. Work in our laboratory focuses on the molecular machineries that mediate these processes, including (1) sorting signals and adaptor proteins that select cargo proteins for packaging into the transport carriers, (2) microtubule motors and organelle adaptors that drive movement of the transport carriers and other organelles through the cytoplasm, and (3) tethering factors that promote fusion of the transport carriers to acceptor compartments. These machineries are studied in the context of different intracellular transport pathways, including endocytosis, recycling to the plasma membrane, retrograde transport from endosomes to the TGN, biogenesis of lysosomes and LROs, and polarized sorting in epithelial cells and neurons. Knowledge gained from this research is applied to the elucidation of disease mechanisms, including congenital disorders of protein traffic such as the pigmentation and bleeding disorder Hermansky-Pudlak syndrome (HPS) and the neurocutaneous disorder MEDNIK syndrome, neurodegenerative disorders such as Alzheimers disease, and the exploitation of intracellular transport by pathogens such as HIV-1. An AP-1/clathrin pathway for the sorting of transmembrane receptors to the somatodendritic domain of hippocampal neurons - A major focus of research in our laboratory is on processes mediated by recognition of sorting signals in the cytosolic tails of transmembrane proteins by adaptor proteins (APs) that are components of protein coats (e.g., clathrin coats). Two types of sorting signal referred to as tyrosine-based and dileucine-based participate in various sorting events, including endocytosis, transport to lysosomes and melanosomes, and sorting to the basolateral surface of polarized epithelial cells. In recent years, we extended our studies to the role of signal-adaptor interactions in the process of polarized sorting in neurons. We found that many of these proteins are sorted to the somatodendritic domain by interaction of tyrosine-based or dileucine-based signals with the adaptor protein complex AP-1. More recently, in collaboration with Morten Nielsen (Aarhus University, Denmark), we found that the intracellular sorting receptor SorLA is sorted to the somatodendritic domain of neurons and the basolateral domain of polarized epithelial cells by virtue of an acidic cluster in its cytosolic tail that interacts with AP-1. Together with previous work, these findings establish the AP-1 complex as a global regulator of polarized sorting in different cell types. Defects in polarized sorting likely underlie the pathogenesis of several neurocutaneous disorders caused by mutation in sigma1 subunit isoforms, such as the MEDNIK syndrome (sigma1A), Fried/Pettigrew syndrome (sigma1B) and pustular psoriasis (sigma1C). Polarized organelle segregation in neurons by differential interactions with microtubule motors - Polarized sorting of newly synthesized proteins to the somatodendritic and axonal domains of neurons occurs by selective incorporation into distinct populations of vesicular transport carriers. We recently found that segregation of these carriers to their corresponding neuronal compartments occurs at a region in the axon hillock named the pre-axonal exclusion zone (PAEZ) though differential coupling to different microtubule motors. We also discovered a chain of interactors including Rab5, the FHF complex and dynein-dynactin that retrieves somatodendritic proteins from the axon, thus contributing to their somatodendritic distribution at steady state. A role for AP-1 is sorting presenilin-2 to late endosomes and lysosomes - In collaboration with Wim Annaert (VIB Center for the Biology of Disease, Leuven, Belgium), we demonstrated an additional role for the AP-1 complex in the sorting of a novel form of gamma-secretase to late endosomes and lysosomes. Gamma-secretases are a family of intramembrane-cleaving proteases involved in the pathogenesis of Alzheimer's disease. We identified a sorting motif in the cytosolic tail of the presenilin-2 (PSEN2) subunit of gamma-secretase that targets this enzyme to late endosomes and lysosomes. This motif is recognized in a phosphorylation-dependent manner by AP-1. PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates a prominent pool of intracellular A that contains longer, more pathogenic amyloid-beta peptide. These findings reveal potentially important roles for lysosome-generated amyloid-beta peptide in Alzheimers disease. Biochemical and functional studies of AP-4 - Another adaptor protein complex named AP-4 is a component of a non-clathrin coat involved in protein sorting at the trans-Golgi network (TGN). Considerable interest in this complex has arisen from the recent discovery that mutations in each of its four subunits are the cause of a congenital intellectual disability and movement disorder in humans. We discovered that an accessory protein named tepsin interacts with AP-4 via interaction of two phylogenetically conserved peptide motifs with the beta4 and epsilon ear domains of AP-4. The bivalency of the interactions increases the avidity of tepsin for AP-4 and may enable cross-linking of multiple AP-4 heterotetramers, thus contributing to the assembly of the AP-4 coat. In addition to revealing critical aspects of this coat, these findings extended the paradigm of peptide-ear interactions, previously established for clathrin-AP-1/AP-2 coats, to a non-clathrin coat. Furthermore, in collaboration with Dennis Drayna (NIDCD, NIH), we identified rare sequence variants of the epsilon subunit of AP-4 that are associated with persistent developmental stuttering. We additionally demonstrated that AP-4 interacts with NAGPA, another protein previously linked to developmental stuttering. These findings implicate deficits in intracellular trafficking in persistent stuttering. Identification of TSSC1 as a novel component of the endosomal retrieval machinery - Endosomes function as a hub for multiple protein sorting events, including retrograde transport to the trans-Golgi network (TGN) and recycling to the plasma membrane. These processes are mediated by tubular-vesicular carriers that bud from early endosomes and fuse with a corresponding acceptor compartment. We previously investigated the role of two multisubunit tethering complexes named GARP and EARP that participate in SNARE-dependent fusion of endosome-derived carriers with the TGN and recycling endosomes, respectively. We have now discovered that a previously uncharacterized WD40/beta-propeller protein named TSSC1 is a specific interactor of both GARP and EARP, and a novel component of the endosomal retrieval machinery. Interference with TSSC1 impairs both retrograde transport to the TGN and retrieval to the plasma membrane. These findings contribute to the understanding of the pathogenesis of progressive cerebello-cerebral atrophy type 2, a neurodegenerative disorder caused by mutations in the shared Vps53 subunit of GARP and EARP.